Abstract. Gram-negative bacteria have caused many of the most persistent infections as well as some of the deadliest pandemics in the world. Some strains have developed resistant to all available drugs, thus leading to increasing mortality from previously treatable bacterial infections. The National Strategy for Combating Antibiotic-Resistant Bacteria released by the White House in September 2014 focused on the need for (i) advancing the development of methods for identification and characterization of bacteria, (ii) accelerating basic research for new antibiotics, and (iii) improving capabilities for surveillance of antibiotic-resistant bacteria. This proposal focuses on the development of innovative tandem mass spectrometry approaches for characterization of lipopolysaccharides (LPS), the primary constituent of the outer membrane of Gram-negative bacteria that protects the membrane from chemical attack and is recognized by the immune system during pathogenic invasion. Structural characterization of LPS is critical to understanding how the structure of LPS influences immune stimulation as well as facilitating development of new antimicrobials and vaccines. This is a significant analytical challenge due to the branched structures and amphipathic properties of LPS. The escalating concerns about antibiotic resistance bacteria and the need for better avenues of defense against infectious diseases have motivated the proposed work. The objectives of this proposal are: Aim 1: Development of ultraviolet photodissociation (UVPD) mass spectrometry via hierarchical, decision-tree workflows for top-down characterization of LPS to facilitate high throughput analysis; Aim 2: Development of MS/MS approaches for serotyping of Gram-negative bacteria based on LPS; Aim 3: Examination of peptide/LPS interactions via native-spray mass spectrometry to provide both mechanistic information and screening capabilities for new antimicrobials, and Aim 4: Applications to a variety of structural problems related to the biosynthesis of LPS in Gram- negative bacteria and characterization of hybrid O-antigen/lipid A molecules in vesicle-based vaccines. We have established a new collaboration with Dr. Bryan Davies' group to develop mass spectrometry methods to characterize peptide/lipid A interactions in support of the hunt for better antimicrobials. The continued collaboration with Dr. Stephen Trent's group emphasizes: (i) approaches for deciphering the biosynthetic pathways of bacterial LPS that endow them with the remarkable ability to re-design their outer membranes and develop antibiotic resistance, and (ii) innovative vaccine design based on antigenic LPS in vesicles.